Composition comprising material for regulating oct4 modification to repress stemness

ABSTRACT

The present invention relates to a composition for repressing the sternness of stem cells, which comprises a material for regulating OCT4 modification. The material for regulating OCT4 modification according to the present invention may regulate the phosphorylation or methylation of OCT4 and inhibit Wnt signaling, thereby effectively reducing the sternness of various stem cells. Therefore, since it can be effectively used in inhibition of proliferation, recurrence and metastasis of cancer, and inhibition of resistance to an anticancer agent, and can reduce sternness even in normal stem cells, it is expected that the time for differentiation of embryonic stem cells into specific cells is shortened, and efficiency is increased.

TECHNICAL FIELD

The present invention relates to a composition comprising a material forregulating OCT4 modification to repress stemness.

BACKGROUND ART

Most anticancer agents which have been actively developed andsubstantially used in cancer treatment in recent years are drugstargeting cancer cells which are rapidly proliferated. According toanticancer treatment using these drugs, in an early stage, it seems thatcancer cells are effectively killed to cure cancer, but eventuallycancer stem cells still remaining in the body are not removed, andrecurrence and/or metastasis of cancer actively happen, resulting infrequently caused problems of resistance to conventional cancer therapy.For this reason, recently, interest in cancer stem cells is increasing.

Cancer stem cells are cancer cells which possess an ability of unlimitedself-renewal similar to normal stem cells but are slowly proliferatedunlike normal cancer cells, and possess distinct abilities of stem cellsto self-renew and differentiate, and they have been known to havedifferent mechanisms from previously known cancer cells. Cancer stemcells, belonging to a specific subpopulation having a potential todifferentiate into various cancer cell groups and self-renewal, exhibitdrug and radiation resistances, resulting in recurrence or metastasis ofcancer as well as initiation of cancer, and therefore inhibition of theproliferation of cancer stem cells is important for cancer treatment(Korean Unexamined Patent Application No. 10-2014-0143989).

In addition, since it is difficult for stem cell therapeutics which havebeen actively studied in recent years to regulate the proliferation orsurvival of stem cells, there are side effects. Typical side effects maybe attributed to carcinogenesis of undifferentiated stem cells remainingin the body after treatment is finished.

Therefore, if there is a method of regulating the stemness of stem cellsincluding cancer stem cells, it can be safely applied to various stemcell applications, and increase cancer therapeutic effects, therebyeffectively inhibiting recurrence and metastasis of cancer andresistance.

Meanwhile, octamer-binding transcription factor 4 (OCT4), also known asPOU5F1 (POU domain, class 5, transcription factor 1), is a transcriptionfactor which is expressed in a conventional embryonic stem cell, servesto prevent cell differentiation and disappears when naturaldifferentiation of cells begins. OCT4 is known as a marker specific forpluripotent embryonic stem cells.

However, in terms of cancer therapy, as conventionally known, beyond theuse of OCT4 as a marker for detecting stemness, the relationship betweenregulation of OCT4 phosphorylation or methylation and regulation of Wntsignaling, such as regulation of stemness by directly regulating OCT4itself by regulating the phosphorylation or methylation of a specificamino acid sequence among the amino acid sequence of OCT4, is not knownyet. In addition, the relationship between regulation of OCT4phosphorylation and Wnt signaling and regulation of stemness, forexample, regulation of stemness by regulating Wnt signaling as well asregulating OCT4 phosphorylation, is not known yet.

DISCLOSURE OF INVENTION Technical Problem

To solve the problems caused by conventional technology, the presentinvention is directed to provide a composition which includes amaterial, as an active ingredient, for repressing the stemness of stemcells and blocking Wnt signaling by regulating OCT4 phosphorylation ormethylation so as to repress the stemness of various stem cells such ascancer stem cells.

However, technical problems to be solved in the present invention arenot limited to the above-described problems, and other problems whichare not described herein will be fully understood by those of ordinaryskill in the art from the following descriptions.

Solution to Problem

To attain the object of the present invention, the present inventionprovides a composition which comprises a material for regulating OCT4modification to repress the stemness of stem cells, wherein the OCT4modification is regulated by any one or more selected from the groupconsisting of the following methods:

a) the phosphorylation of serine 281 in the amino acid sequence of mouseOCT4 or serine 288 in the amino acid sequence of human OCT4;

b) inhibition of the methylation of arginine 179 in the amino acidsequence of mouse OCT4, arginine 186 in the amino acid sequence of humanOCT4, lysine 215 in the amino acid sequence of mouse OCT4, or lysine 222in the amino acid sequence of human OCT4; and

c) inhibition of the phosphorylation of serine 351 in the amino acidsequence of mouse OCT4 or serine 359 in the amino acid sequence of humanOCT4.

According to an exemplary embodiment of the present invention, thecomposition may further comprise a material for blocking the activationof Wnt signaling.

According to another exemplary embodiment of the present invention, theamino acid sequence of the mouse OCT4 may be represented by an aminoacid sequence of SEQ ID NO: 1.

According to still another exemplary embodiment of the presentinvention, the amino acid sequence of the human OCT4 may be representedby an amino acid sequence of SEQ ID NO: 2.

According to yet another exemplary embodiment of the present invention,the material may be any one selected from the group consisting of apeptide, a protein, a nucleotide and a compound.

According to yet another exemplary embodiment of the present invention,the material may comprise a material for inhibiting the expression ofcasein kinase 2 alpha 1 (CSNK2A1).

According to yet another exemplary embodiment of the present invention,the material for inhibiting CSNK2A1 expression may be a short hairpinRNA (shRNA) represented by a base sequence of SEQ ID NO: 5 or 6.

According to yet another exemplary embodiment of the present invention,the stem cells may be any one selected from the group consisting ofembryonic stem cells, gametes, and cancer stem cells.

According to yet another exemplary embodiment of the present invention,the composition may inhibit proliferation, self-renewal, clustering, orsurvival of stem cells.

In addition, the present invention provides a kit for regulating thestemness of stem cells, which comprises a material for regulatingmodification of OCT4 and a material for activating Wnt signaling.

In addition, the present invention provides a cell therapeutic, whichcomprises a material for regulating modification of OCT4 and stem cellsfor cell therapy as active ingredients.

According to an exemplary embodiment of the present invention, the celltherapeutic may further comprise a material for blocking the activationof Wnt signaling.

According to another exemplary embodiment of the present invention, thecell therapeutic may inhibit metastasis, survival or carcinogenesis ofstem cells for cell therapy.

In addition, the present invention provides a method of repressingsternness by regulating OCT4 modification in vitro, the methodcomprising any one or more selected from the group consisting of thefollowing steps:

(a) phosphorylating serine 281 in the amino acid sequence of mouse OCT4or serine 288 in the amino acid sequence of human OCT4;

(b) inhibiting the methylation of arginine 179 in the amino acidsequence of mouse OCT4, arginine 186 in the amino acid sequence of humanOCT4, lysine 215 in the amino acid sequence of mouse OCT4, or lysine 222in the amino acid sequence of human OCT4; and

(c) inhibiting the phosphorylation of serine 351 in the amino acidsequence of mouse OCT4 or serine 359 in the amino acid sequence of humanOCT4.

According to an exemplary embodiment of the present invention, the step(c) may further comprise blocking the activation of Wnt signaling.

In addition, the present invention provides a method of regulatingstemness by regulating OCT4 modification in vitro, the methodcomprising: inhibiting the phosphorylation of serine 351 in the aminoacid sequence of mouse OCT4 or serine 359 in the amino acid sequence ofhuman OCT4; and activating Wnt signaling.

In addition, the present invention provides a pharmaceutical compositionfor preventing or treating cancer, the composition comprising: amaterial for regulating OCT4 modification.

According to an exemplary embodiment of the present invention, thepharmaceutical composition may further comprise an anticancer agent.

According to another exemplary embodiment of the present invention, thepharmaceutical composition may inhibit proliferation, survival,metastasis or recurrence of cancer, or resistance to an anticanceragent.

According to still another exemplary embodiment of the presentinvention, the pharmaceutical composition may further comprise amaterial for blocking the activation of Wnt signaling.

In addition, the present invention provides a method of preventing ortreating cancer, the method comprising: administering the pharmaceuticalcomposition into a subject.

In addition, the present invention provides a use of the pharmaceuticalcomposition for preventing or treating cancer.

In addition, the present invention provides an antibody for detectingwhether serine 351 in the amino acid sequence of mouse OCT4 or serine359 in the amino acid sequence of human OCT4 is phosphorylated, andspecifically, recognizing an epitope represented by an amino acidsequence of SEQ ID NO: 7 or 8 and specifically binding to phosphorylatedserine 351 of mouse OCT4 or phosphorylated serine 359 of human OCT4.

In addition, the present invention provides a method of screening amaterial for repressing the stemness of stem cells, the methodcomprising the following steps:

(a) treating cells expressing mouse OCT4 or human OCT4 with a candidatematerial;

(b) detecting any one or more selected from the group consisting of thephosphorylation of serine 281 in the amino acid sequence of mouse OCT4or serine 288 in the amino acid sequence of human OCT4, the methylationof arginine 179 in the amino acid sequence of mouse OCT4, arginine 186in the amino acid sequence of human OCT4, lysine 215 in the amino acidsequence of mouse OCT4, or lysine 222 in the amino acid sequence ofhuman OCT4, and the phosphorylation of serine 351 in the amino acidsequence of mouse OCT4 or serine 359 in the amino acid sequence of humanOCT4; and

(c) selecting the candidate material as a material for repressing thestemness of stem cells when serine 281 in the amino acid sequence ofmouse OCT4 or serine 288 in the amino acid sequence of human OCT4 isphosphorylated, when the methylation of arginine 179 in the amino acidsequence of mouse OCT4, arginine 186 in the amino acid sequence of humanOCT4, lysine 215 in the amino acid sequence of mouse OCT4, or lysine 222in the amino acid sequence of human OCT4 is inhibited, or when thephosphorylation of serine 351 in the amino acid sequence of mouse OCT4or serine 359 in the amino acid sequence of human OCT4 is inhibited.

According to an exemplary embodiment of the present invention, thescreening method may comprise: detecting whether Wnt signaling isactivated in cells as the step (b); and

selecting the candidate material as a material for repressing thestemness of stem cells when the Wnt signaling is not activated as thestep (c).

In addition, the present invention provides a method of impartinginformation for diagnosing whether the stemness of stem cells isrepressed, the method comprising the following steps:

(a) detecting any one or more selected from the group consisting of thephosphorylation of serine 281 in the amino acid sequence of mouse OCT4or serine 288 in the amino acid sequence of human OCT4, the methylationof arginine 179 in the amino acid sequence of mouse OCT4, arginine 186in the amino acid sequence of human OCT4, lysine 215 in the amino acidsequence of mouse OCT4, or lysine 222 in the amino acid sequence ofhuman OCT4, and the phosphorylation of serine 351 in the amino acidsequence of mouse OCT4 or serine 359 in the amino acid sequence of humanOCT4 from cells expressing mouse OCT4 or human OCT4; and

(b) determining that the stemness of stem cells is repressed when serine281 in the amino acid sequence of mouse OCT4 or serine 288 in the aminoacid sequence of human OCT4 is phosphorylated, when the methylation ofarginine 179 in the amino acid sequence of mouse OCT4, arginine 186 inthe amino acid sequence of human OCT4, lysine 215 in the amino acidsequence of mouse OCT4, or lysine 222 in the amino acid sequence ofhuman OCT4 is inhibited, or when the phosphorylation of serine 351 inthe amino acid sequence of mouse OCT4 or serine 359 in the amino acidsequence of human OCT4 is inhibited.

According to an exemplary embodiment of the present invention, theinformation imparting method may comprise: confirming whether Wntsignaling is activated in cells expressing mouse OCT4 or human OCT4 asthe step (a); and

determining that the stemness of stem cells is repressed when the Wntsignaling is not activated as the step (b).

In addition, the present invention provides a method of diagnosingwhether the stemness of stem cells is repressed, the method comprisingthe following steps:

(a) detecting any one or more selected from the group consisting of thephosphorylation of serine 281 in the amino acid sequence of mouse OCT4or serine 288 in the amino acid sequence of human OCT4, the methylationof arginine 179 in the amino acid sequence of mouse OCT4, arginine 186in the amino acid sequence of human OCT4, lysine 215 in the amino acidsequence of mouse OCT4, or lysine 222 in the amino acid sequence ofhuman OCT4, and the phosphorylation of serine 351 in the amino acidsequence of mouse OCT4 or serine 359 in the amino acid sequence of humanOCT4; and

(b) determining that the stemness of stem cells is repressed when serine281 in the amino acid sequence of mouse OCT4 or serine 288 in the aminoacid sequence of human OCT4 is phosphorylated, when the methylation ofarginine 179 in the amino acid sequence of mouse OCT4, arginine 186 inthe amino acid sequence of human OCT4, lysine 215 in the amino acidsequence of mouse OCT4, or lysine 222 in the amino acid sequence ofhuman OCT4 is inhibited, or when the phosphorylation of serine 351 inthe amino acid sequence of mouse OCT4 or serine 359 in the amino acidsequence of human OCT4 is inhibited.

According to an exemplary embodiment of the present invention, thediagnosing method may comprise:

confirming whether Wnt signaling is activated in cells expressing mouseOCT4 or human OCT4 as the step (a); and

determining that the stemness of stem cells is repressed when the Wntsignaling is not activated as the step (b).

Advantageous Effects of Invention

A material for regulating OCT4 modification according to the presentinvention can regulate the phosphorylation or methylation of OCT4 andinhibit Wnt signaling, thereby effectively reducing the stemness ofvarious stem cells. Therefore, since it can be effectively used ininhibition of proliferation, recurrence and metastasis of cancer, andinhibition of resistance to an anticancer agent, and can reduce stemnesseven in normal stem cells, the time for differentiation of embryonicstem cells into specific cells can be shortened, and efficiency can beincreased. In addition, when the material for regulating OCT4modification according to the present invention is used in cell therapyusing embryonic stem cells, undifferentiated embryonic stem cellsremaining after the therapy can be completely removed, and side effectsgenerated in carcinogenesis can be effectively inhibited. For thisreason, it is expected that the stability of cell therapy usingembryonic stem cells can be significantly increased.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows a result of confirming that mutant OCT4 is expressed in atransformed cell line according to an exemplary embodiment of thepresent invention at an amount similar to wild type (WT).

FIG. 2 shows a result of confirming the effects of inhibition ofphosphorylation and mimetic mutation of serine 281 of OCT4 on theself-renewal ability of stem cells in a transformed cell line accordingto an exemplary embodiment of the present invention with the formationof embryonic stem cell clusters using alkaline phosphatase staining.

FIG. 3 shows an SDS-PAGE result of confirming FLAG-OCT4 isolated fromtransformed embryonic stem cells expressing FLAG-OCT4 according to anexemplary embodiment of the present invention by immunoprecipitationusing an anti-FLAG antibody.

FIG. 4 shows a result of confirming an isolated FLAG-OCT4 proteinaccording to an exemplary embodiment of the present invention using amass spectrometer.

FIG. 5 shows a result of confirming the base sequence of a vectormutated so that it is impossible to methylate methylation sites(arginine 179 and lysine 215) of mouse OCT4 according to an exemplaryembodiment of the present invention.

FIG. 6 shows a result of confirming that mutant OCT4 is expressed in atransformed cell line according to an exemplary embodiment of thepresent invention at an amount similar to the wild type (WT).

FIG. 7 shows a result of confirming an effect of an OCT4methylation-inhibited mutant on the self-renewal ability of stem cellsin a transformed cell line according to an exemplary embodiment of thepresent invention with the formation of embryonic stem cell clustersusing alkaline phosphatase staining.

FIG. 8 shows sequences of 14 amino acids including serine at the 351position for designing OCT4 phosphorylation-specific antibodiesaccording to an exemplary embodiment of the present invention.

FIG. 9 shows a result of verifying an OCT4 phosphorylated antibodyaccording to an exemplary embodiment of the present invention byrecognizing a peptide at a corresponding phosphorylated site with anantibody during western blotting.

FIG. 10 shows a result of verifying whether OCT4 phosphorylation isrecognized when resistant OCT4 expression is inhibited by treating anembryonic stem cell line according to an exemplary embodiment of thepresent invention with doxycycline.

FIG. 11 shows a result of verifying a phosphorylated antibody in anembryonic stem cell line according to an exemplary embodiment of thepresent invention using immunofluorescence.

FIG. 12 shows a result of verifying a phosphorylated OCT4 antibodyaccording to an exemplary embodiment of the present invention throughimmunoprecipitation.

FIG. 13 shows a result of confirming whether OCT4 phosphorylation isrecognized in human cancer cells according to an exemplary embodiment ofthe present invention.

FIG. 14 shows a result of confirming that mutant OCT4 is expressed in atransformed cell line according to an exemplary embodiment of thepresent invention at a quantity similar to the wild type (WT).

FIG. 15 shows a result of confirming effects of an OCT4-serine 351phosphorylation-inhibited mutant (S351A) and an OCT4-serine 351phosphorylation-mimetic mutant (S351E) on self-renewal ability of stemcells in a transformed cell line according to an exemplary embodiment ofthe present invention with the formation of embryonic stem cell clustersusing alkaline phosphatase staining.

FIG. 16 shows a result of confirming a transduced embryonic stem cellline according to an exemplary embodiment of the present invention usingwestern blotting.

FIG. 17 shows a result of confirming the self-renewal ability of stemcells in a transduced embryonic stem cell line according to an exemplaryembodiment of the present invention using alkaline phosphatase staining.

FIG. 18 shows a result of confirming self-renewal ability of stem cellsin a transduced embryonic stem cell line according to an exemplaryembodiment of the present invention with a difference in SSEA-1expression.

FIG. 19 shows a result of analyzing a gene expression pattern and ChIPdata of a transduced embryonic stem cell line according to an exemplaryembodiment of the present invention.

FIG. 20 shows the result of FIG. 19 according to an exemplary embodimentof the present invention, visualized by analyzing signaling pathways.

FIG. 21 shows a result of amplification analysis of a gene set inresponse to Wnt signaling by the pathway analysis of FIG. 20 accordingto an exemplary embodiment of the present invention.

FIG. 22 shows a result of confirming the self-renewal ability of stemcells when Wnt signaling is artificially activated in a transducedembryonic stem cell line according to an exemplary embodiment of thepresent invention using alkaline phosphatase staining.

FIG. 23 shows a result of confirming the self-renewal ability of stemcells when Wnt signaling is artificially activated in a transducedembryonic stem cell line according to an exemplary embodiment of thepresent invention by SSEA-1 expression.

FIG. 24 shows a result of confirming that a kinase inducing OCT4phosphorylation according to an exemplary embodiment of the presentinvention is casein kinase 2 alpha 1 (CSNK2A1).

FIG. 25 shows that CSNK2A1 as an in vitro kinase according to anexemplary embodiment of the present invention induces OCT4phosphorylation.

FIG. 26 schematically shows that the self-renewal ability of stem cellsis regulated by regulation of OCT4 modification (post-translationalmodification) according to an exemplary embodiment of the presentinvention.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention provides a composition for repressing thesternness of stem cells, which comprises a material for regulating OCT4modification, wherein

the OCT4 modification is regulated by any one or more selected from thegroup consisting of the following methods:

a) the phosphorylation of serine 281 in the amino acid sequence of mouseOCT4 or serine 288 in the amino acid sequence of human OCT4;

b) inhibition of the methylation of arginine 179 in the amino acidsequence of mouse OCT4, arginine 186 in the amino acid sequence of humanOCT4, lysine 215 in the amino acid sequence of mouse OCT4, or lysine 222in the amino acid sequence of human OCT4; and

c) inhibition of the phosphorylation of serine 351 in the amino acidsequence of mouse OCT4 or serine 359 in the amino acid sequence of humanOCT4.

According to the present invention, the composition may further comprisea material for blocking the activation of Wnt signaling.

According to the present invention, an amino acid sequence of the mouseOCT4 may be represented by an amino acid sequence of SEQ ID NO: 1 (NCBIReference Sequence: NP_038661.2), and an amino acid sequence of thehuman OCT4 may be represented by an amino acid sequence of SEQ ID NO: 2(NCBI Reference Sequence: NP_002692.2).

According to the present invention, the amino acid sequence of SEQ IDNO: 1 may be encoded by a base sequence of SEQ ID NO: 3 (NCBI ReferenceSequence: NM_013633.3).

According to the present invention, the amino acid sequence of SEQ IDNO: 2 may be encoded by a base sequence of SEQ ID NO: 4 (NCBI ReferenceSequence: NM_002701.5).

According to the present invention, the material may be any one selectedfrom the group consisting of a peptide, a protein, a nucleotide and acompound, and preferably a material for inhibiting CSNK2A1 expression,and the material for inhibiting CSNK2A1 expression may include a shorthairpin RNA (shRNA) represented by a base sequence of SEQ ID NO: 5 or 6,and may be any material capable of regulating OCT4 modification withoutlimitation.

The term “OCT4” used herein is a member of the family of POUtranscription factors and expressed in pluripotent embryonic stem cellsand gametes. Members of the POU transcription factor family haveconserved DNA-binding domains, that is, POU domains, and the domainswere first identified in Pit-1, Oct-1, Oct-2 and Unc-86 among thetranscription factors. OCT4 activates transcription by an octamer motifwhich is adjacent to or spaced apart from a transcription initiationsite.

The term “Wnt” used herein is a secreted glycoprotein, and a ligand fora 7-transmembrane receptor, Frizzled, and a conjugated receptor,1-transmembrane lowdensity lipid protein receptor-associated protein 5,6(LRPS/6). The Wnt gene family encodes a large set of secreted proteinsassociated with Intl/Wnt1 proto-oncogenes and Drosophila wingless (Wg),which is a homologous gene of Drosophila Wnt1. Wnt is expressed invarious tissues and organs, and plays a pivotal role in segmentation indrosophila; endodermal development in C. elegans; and a variety ofdevelopment processes including establishment of limb polarity, neuraldifferentiation, kidney morphogenesis, sex determination and braindevelopment in mammals. The Wnt signaling pathway is conserved inspecies ranging from nematoda, drosophila to mammals, and an importantregulator for animal development in embryonic development and matureorganisms.

The term “stem cells” used herein is the generic term forundifferentiated cells having an ability to differentiate into varioustypes of tissue cells, that is, stemness. These stem cells are largelydivided into embryonic stem cells, which can be produced using anembryo, adult stem cells, gametes, and cancer stem cells, and theembryonic stem cells refer to the stage of a cell mass before a specificorgan only aged less than 14 days after fertilization is formed, andrecently, the embryonic stem cells are also produced from normal cellsthrough reprogramming. Accordingly, the stem cell is any cell that candifferentiate into all cells and tissues constituting a body withoutlimitation. Adult stem cells are extracted from umbilical cord blood,bone marrow, blood, etc., and refer to primitive cells just beforedifferentiating into cells of a specific organ such as bone, liver,blood or the like. Gametes are cells that transfer genetic informationto the next generation through reproduction, and include sperm and eggsin humans, but the present invention is not limited thereto. Inaddition, the stem cells are cells that can maintain a new stem cell ina cluster through self-renewal in a process of producing a cell clusterby forming clones, and have an ability to form one or more types ofspecific cells through differentiation.

The term “stemness” used herein refers to the characteristic ability ofstem cells, which includes multipotency and unipotency as well aspluripotency which can differentiate into all types of cellsconstituting an organism such as nerves, blood, cartilage, etc., and thestemness also includes an ability to self-replicate or self-renew. Italso means that stem cells have an ability to form a cluster throughself-replication or self-renewal. In this case, the stemness may includean ability to differentiate into various types of cells from embryonicstem cells obtained from an embryo generated from a fertilized egg, andwhile there is a difference in the functional aspect, also include anability to differentiate into various types of cells from adult stemcells conserved in each tissue of an adult body.

Cancer stem cells generically refer to cancer cells having stemness,which is the distinct ability of stem cells, such as self-renewal ordifferentiation. Cancer stem cells, unlike general cancer cells, aregenerally grown slowly or maintained in a dormant state, such that theyhave resistance to an anticancer agent under normal tumor growthconditions (refers to a state without cell stress since there aresufficient nutrients (glucose) required for cell growth and an abundanttumor microenvironment), and for example, the cancer stem cells may bedifferent from general cancer cells in terms of the function of a keymaterial for regulating metabolism since expression of atranscription-regulating factor such as PGC-1α is controlled unlikenormal tumor cells. Cancer stem cells encompass cells havinginfiltrating and metastatic abilities, acquiring resistance to apoptosisin a nutrient-deficient state through such differentmetabolism-regulating abilities and regulation of cell signaltransduction pathways metabolically associated therewith. However, thereis no limitation as long as cells can differentiate into general cancercells.

In the present invention, the stem cells may be any one selected fromthe group consisting of embryonic stem cells, gametes, and cancer stemcells, but there is no limitation as long as cells are a type of stemcells expressing an OCT4 protein.

In the present invention, the composition may repress the stemness ofstem cells, such that proliferation, self-renewal, a clustering abilityor survival of stem cells can be inhibited, and therefore thecomposition can be used in all fields using regulation of stem cells.

In addition, the present invention provides a kit for regulating thestemness of stem cells, which comprises a material for regulating OCT4modification and a material for activating Wnt signaling.

Here, the kit comprising a material for regulating OCT4 modification anda material for activating Wnt signaling may regulate the stemness ofstem cells by repressing the stemness by inhibiting the activation ofWnt signaling by inhibiting the phosphorylation of serine 351 in theamino acid sequence of mouse OCT4 or serine 359 in the amino acidsequence of human OCT4 by an OCT4 modification-regulating material, andby increasing the stemness again by activating only Wnt signaling by thematerial for activating Wnt signaling in the OCT4phosphorylation-inhibited cells. To regulate stemness as describedabove, each of the OCT4 modification-regulating material and thematerial for activating Wnt signaling may be applied one or more timeswithout limitation, and there is no limitation to the order of applyingthese materials, and thus, the materials may be applied simultaneouslyor separately.

In the present invention, the kit may include a container; a protocol; amaterial for regulating OCT4 modification; and a material for activatingWnt signaling. The container may serve to package each of the materialfor regulating OCT4 modification and the material for activating Wntsignaling, and to store and fix these materials. The container may beformed of, for example, plastic or glass, but the present invention isnot limited thereto. In addition, the protocol included in the kit forregulating the stemness of stem cells according to the present inventionmay contain instructions for regulating the stemness of stem cells. Theinstructions may be described in a separate sheet or booklet, other thanthe container, and the sheet or booklet may be included in addition tothe material for regulating OCT4 modification and the material foractivating Wnt signaling in the container.

In addition, the present invention provides a cell therapeutic, whichcomprises the material for regulating OCT4 modification and stem cellsfor cell therapy as active ingredients.

In the present invention, the cell therapeutic may further comprise amaterial for blocking the activation of Wnt signaling.

The term “cell therapeutic” used herein refers to a drug used intreatment, diagnosis and prevention by a series of methods forproliferating or selecting autologous, allogenic or xenogenic livingcells in vitro to restore the function of cells or tissue, or changingbiological characteristics of cells by different methods.

In the present invention, the cell therapeutic may regulate stem cellsby inhibiting metastasis or survival of therapeutic stem cells, orpreventing cancer caused by carcinogenesis of therapeutic stem cellsremaining after treatment. However, there is no limitation as long as acell therapeutic has an effect induced by inhibition of the OCT4function.

In addition, the present invention provides a method of repressingstemness by regulating OCT4 modification in vitro, which comprises anyone or more selected from the group consisting of the following steps:

(a) the phosphorylation of serine 281 in the amino acid sequence ofmouse OCT4 or serine 288 in the amino acid sequence of human OCT4;

(b) inhibition of the methylation of arginine 179 in the amino acidsequence of mouse OCT4, arginine 186 in the amino acid sequence of humanOCT4, lysine 215 in the amino acid sequence of mouse OCT4, or lysine 222in the amino acid sequence of human OCT4; and

(c) inhibition of the phosphorylation of serine 351 in the amino acidsequence of mouse OCT4 or serine 359 in the amino acid sequence of humanOCT4.

In the present invention, the step (c) may further comprise blocking theactivation of Wnt signaling.

In addition, the present invention provides a method of regulatingstemness by regulating OCT4 modification in vitro, which comprises:inhibiting the phosphorylation of serine 351 in the amino acid sequenceof mouse OCT4 or serine 359 in the amino acid sequence of human OCT4;and activating Wnt signaling.

In addition, the present invention provides a pharmaceutical compositionfor preventing or treating cancer, which comprises the material forregulating OCT4 modification.

In addition, the present invention provides a method of preventing ortreating cancer, which comprises administering the pharmaceuticalcomposition into a subject.

In addition, the present invention provides a use of the pharmaceuticalcomposition for preventing or treating cancer.

The term “cancer” used herein refers to the generic term for diseasescaused by aggressive cells which ignore the limit to normal growth ofcells, divided and grown, invasive cells which infiltrate intosurrounding tissues, and metastatic cells which spread to other sites inthe body.

The main causes of cancerous death are metastasis of cancer andtherapeutic resistance, and recently, it has been strictly proven thatits fundamental cause is cancer cells having stemness. Therefore, it isexpected that an anticancer strategy for reducing stemness of cancercells leads to a substantial decrease in death caused by cancer.

There are two major pathways in which cancer cells acquire stemness: oneis carcinogenesis of normal stem cells; and the other is reprogrammingof cancer cells. It has been reported that, in cancer patients,particularly, key factors of embryonic stem cells play an important rolein maintaining the stemness of cancer cells, and as malignancy of cancerprogresses, expression of an embryonic stem cell-related gene family,rather than normal tissue stem cell-related genes, is increased.

The term “modification” used herein refers that a specific moiety bindsto a residue of an amino acid after protein synthesis (the process oftranslating mRNA transcribed from DNA and synthesizing a primary chainof amino acids). In the present invention, the protein modificationmoiety may be selected from the group consisting of acylation,alkylation, amidation, butyrylation, carboxylation, glycosylation,formylation, hydroxylation, iodination, oxidation, phosphorylation,propionylation, succinylation, sulfation, glycation, carbonylation,ubiquitination, sumoylation, neddylation and pupylation moieties. Inaddition, the alkylation can be any chemical reaction for producing anatomic group from which one hydrogen atom is removed from an aliphaticsaturated hydrocarbon, selected from the group consisting ofmethylation, acetylation, ethylation, propylation, amylation,hexylation, heptylation, octylation, nonylation and decylation. In thepresent invention, the modification moiety may be a phosphorylation ormethylation moiety, but the present invention is not limited thereto.

The term “phosphorylation” used herein is a biochemical reaction whichadds a phosphate group to a serine (S), threonine (T) or tyrosine (Y)residue of a specific protein, which is catalyzed by a protein kinase.Phosphorylation conventionally changes the function of a target proteinto regulate activity of the protein. As a part of the mechanisms of cellhomeostasis, phosphorylation is merely a temporary process, and reversedby other enzymes called phosphatases. Any unusual events in an aspect ofthe reaction (phosphorylation vs. dephosphorylation) may destroy a cellfunction.

The term “methylation” used herein is broadly classified into DNAmethylation and histone methylation. DNA methylation is binding of amethyl group to the carbon no. 5 of a cytosine base in the nucleotide5′-CpG-3′, and uses a DNA methyltransferase as an enzyme. Meanwhile,histone methylation is binding of a methyl group to a residue of anamino acid, such as arginine (R) or lysine (K), of histone H3 or histoneH4. Histone methylation inhibits or activates transcription depending onwhether methylation occurs at any residue of arginine or lysine. Thearginine residue may be methylated with monomethyl arginine ordimethylarginine by peptidyl arginine methyltransferase (PRMT), in whichthe dimethylarginine may have an asymmetric structure in which twomethyl groups bind to one nitrogen atom or a symmetric structure inwhich a methyl group binds to each of two nitrogen atoms. Even threemethyl groups may bind to a lysine residue by lysine methyltransferase.

According to an exemplary embodiment of the present invention, as aresult of confirming the relationship between the phosphorylation ofserine 281 of mouse OCT4 and OCT4 activation, it is confirmed that thenumber of embryonic stem cells forming clusters is increased in aOCT4-serine 281 phosphorylation-inhibited mutant cell line (S281A),compared with a wild type, whereas the number of clusters is decreasedin a OCT4-serine 281 phosphorylation-mimetic mutant cell line (S281E),compared with the wild type, which shows that, when serine 281 of mouseOCT4 (serine 288 of human OCT4) is phosphorylated, stemness is reduced(refer to Example 1).

According to another exemplary embodiment of the present invention, toconfirm a new methylation site of OCT4, an OCT4 protein is analyzedusing a mass spectrometer after being isolated and purified from mouseembryonic stem cells, and thereby it is confirmed that new methylationoccurs at arginine 179 of mouse OCT4 (arginine 186 of human OCT4) andlysine 215 of mouse OCT4 (lysine 222 of human OCT4), which werepreviously unknown (refer to Example 2-1).

According to still another exemplary embodiment of the presentinvention, as a result of confirming the relationship between OCT4methylation and OCT4 activation, it is confirmed that the number ofembryonic stem cells forming a cluster is decreased in OCT4methylation-inhibited mutant cell lines (R179K and K215R), compared witha wild type, confirming that, when the methylation of mouse arginine 179(arginine 186 of human OCT4) or lysine 215 of mouse OCT4 (lysine 222 ofhuman OCT4) is inhibited, stemness is reduced (refer to Example 2-2).

According to yet another exemplary embodiment of the present invention,it is confirmed that an antibody specifically recognizing thephosphorylation of serine 351 of OCT4 is produced (refer to Example3-1), and the phosphorylation of serine 351 of mouse OCT4 is alsodetected in human cancer cells (NCCIT and NTERA2) (refer to Example3-2).

According to yet another exemplary embodiment of the present invention,as a result of confirming the relationship between the phosphorylationof serine 351 of OCT4 and OCT4 activation, it is confirmed that thenumber of embryonic stem cells forming clusters is decreased in aOCT4-serine 351 phosphorylation-inhibited mutant cell line (S351A),compared with a wild type, whereas the number of clusters is increasedin a OCT4-serine 351 phosphorylation-mimetic mutant cell line (S351E),compared with the wild type, which shows that, when the phosphorylationof mouse serine 351 of OCT4 (serine 359 of human OCT4) is inhibited,stemness is reduced (refer to Example 3-3).

Accordingly, the OCT4 modification-regulating material according to thepresent invention inhibits the function of OCT4 through thephosphorylation of serine 281 of mouse OCT4 or serine 288 of human OCT4;inhibition of the methylation of arginine 179 of mouse OCT4, arginine186 of human OCT4, lysine 215 of mouse OCT4, or lysine 222 of humanOCT4; or inhibition of the phosphorylation of serine 351 of mouse OCT4or serine 359 of human OCT4, such that normal stem cells lose sternnessand become differentiated, cancer cells lose sternness, making itimpossible to survive for a long time, and cancer does not recur afteranticancer therapy.

According to yet another exemplary embodiment of the present invention,as a result of confirming the relationship between OCT4 phosphorylationand regulation of Wnt signaling, it is confirmed that, whenphosphorylation-inhibited mutant (S351A) OCT4 is expressed, Wntsignaling is inhibited, and when Wnt signaling is artificially activatedin cells stably expressing phosphorylation-inhibited mutant OCT4, theself-renewal ability of stem cells is restored (refer to Example 4).

According to yet another exemplary embodiment of the present invention,as a result of confirming a kinase inducing the phosphorylation ofserine 351 of OCT4, it is confirmed that CSNK2A1 induces thephosphorylation of serine 351 of OCT4 (refer to Example 5).

In the present invention, the cancer may be OCT4-associated cancer,preferably including thyroid cancer, cervical cancer, brain cancer, lungcancer, ovarian cancer, liver cancer, pancreatic cancer, prostatecancer, skin cancer, tongue cancer, breast cancer, uterine cancer,gastric cancer, rectal cancer, colorectal cancer or hematologicalcancer, and more preferably including gastric cancer, colorectal cancer,lung cancer, liver cancer, prostate cancer, breast cancer, cervicalcancer or ovarian cancer, but the present invention is not limitedthereto.

The term “prevention” used herein refers to all actions for inhibiting adisease such as cancer or delaying the onset thereof by administrationof the pharmaceutical composition according to the present invention.

The term “treatment” used herein refers to all actions involved inalleviating or beneficially changing symptoms of a disease such ascancer by administration of the pharmaceutical composition according tothe present invention.

The term “administration” used herein refers to providing thecomposition of the present invention to a subject by a suitable method.

The term “subject” used herein refers to a subject in need of treatmentand to which the composition of the present invention may beadministered, and more specifically, a mammal such as a human, or anon-human primate, a mouse, a dog, a cat, a horse or a cow.

The term “pharmaceutical composition” used herein is prepared for thepurpose of prevention or treatment of cancer, and may be used bypreparation in various formulations according to a conventional method.For example, the pharmaceutical composition may be prepared in an oralformulation such as a powder, a granule, a tablet, a capsule, asuspension, an emulsion or a syrup, a medication for topical use such asa cream, a gel, a patch, a spray, an ointment, a plaster, a lotion, aliniment, a paste or a cataplasma, a suppository or a sterilizedinjection.

The pharmaceutical composition according to the present invention mayfurther include a suitable carrier, an excipient or a diluent, which areconventionally used in preparation of a pharmaceutical composition.Here, examples of a carrier, excipient and diluent which can be includedin the composition may include lactose, dextrose, sucrose, anoligosaccharide, sorbitol, mannitol, xylitol, erythritol, maltitol,starch, acacia gum, alginate, gelatin, calcium phosphate, calciumsilicate, cellulose, methyl cellulose, microcrystalline cellulose,polyvinyl pyrrolidone, water, methylhydroxybenzoate,propylhydroxybenzoate, talc, magnesium stearate and mineral oil. Inpreparation, the composition is formulated using a diluent or anexcipient such as a filler, a thickening agent, a binder, a wettingagent, a disintegrant, a surfactant, which are generally used. A solidformulation for oral administration may be a tablet, pill, powder,granule or capsule, and such a solid formulation may be prepared bymixing at least one of excipients, for example, starch, calciumcarbonate, sucrose, lactose and gelatin with the extract. Also, inaddition to the simple excipient, lubricants such as magnesium stearateand talc may also be used. As a liquid formulation for oraladministration, a suspension, a liquid for internal use, an emulsion, ora syrup may be used, and a generally-used simple diluent such as wateror liquid paraffin, as well as various types of excipients, for example,a wetting agent, a sweetener, a fragrance and a preservative may beincluded. A formulation for parenteral administration includes asterilized aqueous solution, a non-aqueous solvent, a suspension, anemulsion, a lyophilizing agent and a suppository. As the non-aqueoussolvent or suspension, propylene glycol, polyethylene glycol, avegetable oil such as olive oil, or an injectable ester such as ethyloleate may be used. As a suppository base, Witepsol, Tween 61, cacaobutter, laurin fat or glycerogelatin may be used.

The pharmaceutical composition of the present invention may beadministered orally or parenterally (e.g., intravenously,subcutaneously, intraperitoneally or locally) depending on a desiredmethod, and a dose of the pharmaceutical composition may vary dependingon the condition and body weight of a patient, the severity of adisease, a drug type, an administration route and time, and may besuitably selected by one of ordinary skill in the art.

The pharmaceutical composition of the present invention is administeredat a pharmaceutically effective amount. The “pharmaceutically effectiveamount” used herein refers to an amount sufficient for treating adisease at a reasonable benefit/risk ratio applicable for medicaltreatment, and an effective dosage may be determined by parametersincluding a type of a patient's disease, severity, drug activity,sensitivity to a drug, administration time, an administration route andan excretion rate, the duration of treatment and drugs simultaneouslyused, and other parameters well known in medical fields. Thepharmaceutical composition of the present invention may be administeredseparately or in combination with other therapeutic agents, and may beadministered in single or multiple doses. In consideration of all of theabove-mentioned parameters, it is important to achieve the maximumeffect with the minimum dose without a side effect, and such a dose maybe easily determined by one of ordinary skill in the art. Administrationmay be performed one or several times per day.

In the present invention, the pharmaceutical composition may furthercomprise an anticancer agent, which is preferably doxorubicin,cisplatin, gemcitabine, oxaliplatin or 5-FU, and the compound is notlimited as long as it can be used as an anticancer agent.

In the present invention, the pharmaceutical composition may repress thestemness of cancer stem cells, thereby inhibiting proliferation,survival, metastasis or recurrence of cancer or resistance to ananticancer agent, but the pharmaceutical composition is not limited aslong as it can exhibit an effect attributed to the repression of thestemness of cancer stem cells.

In the present invention, the pharmaceutical composition may furthercomprise a material for blocking the activation of Wnt signaling.

In addition, the present invention provides an antibody for detectingthe phosphorylation of serine 351 in the amino acid sequence of mouseOCT4 or serine 359 in the amino acid sequence of human OCT4, whichrecognizes an epitope represented by an amino acid sequence of SEQ IDNO: 7 or 8 and specifically binds to phosphorylated serine 351 of mouseOCT4 or phosphorylated serine 359 of human OCT4.

The term “antibody” used herein refers to a polypeptide including animmunoglobulin gene specifically binding to and recognizing an antibodyor a framework region derived from a fragment thereof. The recognizedimmunoglobulin gene includes numerous immunoglobulin variable domaingenes, in addition to κ, λ, α, γ, δ, ε and μ constant domain genes. Alight chain is classified as κ or λ, and a heavy chain is classified asγ, μ, α, δ, or ε, designating an immunoglobulin as IgG, IgM, IgA, IgD orIgE. Typically, an antigen-binding site on an antibody is the mostcritical in terms of binding specificity and affinity. In someembodiments, an antibody or an antibody fragment may be derived from adifferent organism such as a human, a mouse, a rat, a hamster, a rabbit,a camel, etc.

In the present invention, an antibody for detecting the phosphorylationof serine 351 in the amino acid sequence of mouse OCT4 or serine 359 inthe amino acid sequence of human OCT4 may be a polyclonal antibodyderived from a rabbit, but the present invention is not limited thereto,and an epitope recognized by the antibody may be represented by theamino acid sequence of SEQ ID NO: 7 or 8.

In addition, the present invention provides a method of screening amaterial for repressing the stemness of stem cells, which comprises thefollowing steps:

(a) treating cells expressing mouse OCT4 or human OCT4 with a candidatematerial;

(b) detecting any one or more selected from the group consisting of thephosphorylation of serine 281 in the amino acid sequence of mouse OCT4or serine 288 in the amino acid sequence of human OCT4, the methylationof arginine 179 in the amino acid sequence of mouse OCT4, arginine 186in the amino acid sequence of human OCT4, lysine 215 in the amino acidsequence of mouse OCT4, or lysine 222 in the amino acid sequence ofhuman OCT4, and the phosphorylation of serine 351 in the amino acidsequence of mouse OCT4 or serine 359 in the amino acid sequence of humanOCT4; and

(c) selecting the candidate material, as a material for repressing thestemness of stem cells, when serine 281 in the amino acid sequence ofmouse OCT4 or serine 288 in the amino acid sequence of human OCT4 isphosphorylated, when the methylation of arginine 179 in the amino acidsequence of mouse OCT4, arginine 186 in the amino acid sequence of humanOCT4, lysine 215 in the amino acid sequence of mouse OCT4, or lysine 222in the amino acid sequence of human OCT4 is inhibited, or when thephosphorylation of serine 351 in the amino acid sequence of mouse OCT4or serine 359 in the amino acid sequence of human OCT4 is inhibited.

In the present invention, the screening method may comprise detectingwhether Wnt signaling is activated in cells as the step (b); andselecting the candidate material as a material for repressing thestemness of stem cells when the Wnt signaling is not activated as thestep (c).

In addition, the present invention provides a method of screening amaterial for screening a material for repressing the stemness of stemcells, which comprises the following steps:

(a) treating cells expressing mouse OCT4 or human OCT4 with a candidatematerial;

(b) confirming whether Wnt signaling is activated in the cells; and

(c) selecting the candidate material as a material for repressing thestemness of stem cells when Wnt signaling is not activated in the step(b).

In the present invention, the candidate material may be a nucleotide,DNA, RNA, an amino acid, an aptamer, a protein, a compound, a naturalsubstance or a natural extract, and the candidate material is notlimited as long as it can be used for the repression of the stemness ofstem cells.

In addition, the present invention provides a method of impartinginformation for diagnosing whether the stemness of stem cells isrepressed, the method comprising the following steps:

(a) detecting any one or more selected from the group consisting of thephosphorylation of serine 281 in the amino acid sequence of mouse OCT4or serine 288 in the amino acid sequence of human OCT4, the methylationof arginine 179 in the amino acid sequence of mouse OCT4, arginine 186in the amino acid sequence of human OCT4, lysine 215 in the amino acidsequence of mouse OCT4, or lysine 222 in the amino acid sequence ofhuman OCT4, and the phosphorylation of serine 351 in the amino acidsequence of mouse OCT4 or serine 359 in the amino acid sequence of humanOCT4 from cells expressing mouse OCT4 or human OCT4; and

(b) determining that the sternness of stem cells is repressed whenserine 281 in the amino acid sequence of mouse OCT4 or serine 288 in theamino acid sequence of human OCT4 is phosphorylated, when themethylation of arginine 179 in the amino acid sequence of mouse OCT4,arginine 186 in the amino acid sequence of human OCT4, lysine 215 in theamino acid sequence of mouse OCT4, or lysine 222 in the amino acidsequence of human OCT4 is inhibited, or when the phosphorylation ofserine 351 in the amino acid sequence of mouse OCT4 or serine 359 in theamino acid sequence of human OCT4 is inhibited.

In the present invention, the method of imparting information maycomprise:

confirming whether Wnt signaling is activated in cells expressing mouseOCT4 or human OCT4 as the step (a); and

determining that the stemness of stem cells is repressed when the Wntsignal is not activated as the step (b).

In addition, the present invention provides a method of impartinginformation for diagnosing whether the sternness of stem cells isrepressed, the method comprising the following steps:

(a) confirming whether Wnt signaling is activated in cells expressingmouse OCT4 or human OCT4; and

(b) determining that the sternness of stem cells is repressed when theWnt signal is not activated in the step (a).

In addition, the present invention provides a method of determiningwhether the stemness of stem cells is repressed, the method comprisingthe following steps:

(a) detecting any one or more selected from the group consisting of thephosphorylation of serine 281 in the amino acid sequence of mouse OCT4or serine 288 in the amino acid sequence of human OCT4, the methylationof arginine 179 in the amino acid sequence of mouse OCT4, arginine 186in the amino acid sequence of human OCT4, lysine 215 in the amino acidsequence of mouse OCT4, or lysine 222 in the amino acid sequence ofhuman OCT4, and the phosphorylation of serine 351 in the amino acidsequence of mouse OCT4 or serine 359 in the amino acid sequence of humanOCT4 from cells expressing mouse OCT4 or human OCT4; and

(b) determining that the sternness of stem cells is inhibited whenserine 281 in the amino acid sequence of mouse OCT4 or serine 288 in theamino acid sequence of human OCT4 is phosphorylated, when themethylation of arginine 179 in the amino acid sequence of mouse OCT4,arginine 186 in the amino acid sequence of human OCT4, lysine 215 in theamino acid sequence of mouse OCT4, or lysine 222 in the amino acidsequence of human OCT4 is inhibited, or when the phosphorylation ofserine 351 in the amino acid sequence of mouse OCT4 or serine 359 in theamino acid sequence of human OCT4 is inhibited.

In the present invention, the determining method may comprise:

confirming whether Wnt signaling is activated in cells expressing mouseOCT4 or human OCT4 as the step (a); and

determining that the stemness of stem cells is repressed when the Wntsignaling is not activated as the step (b).

In addition, the present invention provides a method of diagnosingwhether the stemness of stem cells is repressed, the method comprisingthe following steps:

(a) confirming whether Wnt signaling is activated in cells expressingmouse OCT4 or human OCT4; and

(b) determining that the stemness of stem cells is repressed when theWnt signaling is not activated in the step (a).

MODE FOR THE INVENTION Examples

Hereinafter, to help in understanding the present invention, exemplaryexamples will be suggested. However, the following examples are merelyprovided to more easily understand the present invention, and not tolimit the present invention.

Example 1. Confirmation of Relationship Between Phosphorylation ofSerine 281 of Mouse OCT4 and OCT4 Activation

The relationship between OCT4 phosphorylation and OCT4 activation may beconfirmed by a method of evaluating maintenance of embryonic stem cells.To perform the evaluation, first, a phosphorylation-inhibited mutantS281A in which serine 281 is replaced with alanine in OCT4 and aphosphorylation-mimetic mutant S281E in which serine 281 is replacedwith glutamic acid in OCT4 were produced through site-directedmutagenesis. Afterward, OCT4 in the embryonic stem cells was removed,and then new cell lines expressing normal OCT4 or OCT4 mutants wereproduced. For cell production, a retrovirus vector expressing wild typeOCT4 and a puromycin-resistant gene, or OCT4 substituted with OCT4phosphorylation-inhibited/mimetic S281A/S281E and a puromycin-resistantgene was produced using a pMSCV-FLAG-puro vector, and then introduced,for transfection, into a mouse embryonic stem cell line ZHBTc4, whichcan inhibit the expression of endogenous OCT4 by treatment withdoxycycline. And then, a medium was treated with puromycin to isolate anormally transfected cell line, and the isolated cell line was treatedwith doxycycline to inhibit the expression of endogenous OCT4 and toallow the substituted exogenous OCT4 protein to be expressed. Afterward,to confirm whether the cells were transfected with the same retrovirus,a mouse fibroblast cell line NIH3T3 in which OCT4 was not expressed wasalso transfected. The protein expression was confirmed through westernblotting with OCT4 antibody (Santa Cruz), and as a control, ACTBantibody (Abcam) was used.

As a result, as shown in FIG. 1, it was demonstrated that OCT4substituted with phosphorylation-inhibited/mimetic S281A/S281E wasexpressed at a similar level to the wild type (WT) in the NIH3T3 cellline.

In addition, before treatment with doxycycline, the cell line was washedwith phosphate buffered saline (PBS), cell clusters were dissociatedinto single cells using trypsin-EDTA (TE) and then a cell culture mediumwas added so that the cell lysate had a final volume of 1 mL. Afterward,the cell lysate was dispensed into a 6-well plate at 100 μL, that is,1/10 of the total volume of the cell lysate, treated with doxycyclineand puromycin, and cultured at 37° C. in 5% CO₂ for 5 to 7 days. Thecultured cells were stained using an alkaline phosphatase assay kit(Medsource Ozone Biomedicals), and observed using a microscope.

As a result, as shown in FIG. 2, it was demonstrated that the OCT4phosphorylation-inhibited mutant cell line (S281A) shows an increase inthe number of embryonic stem cells forming clusters, compared with thewild-type cell line as the control, whereas the OCT4phosphorylation-mimetic mutant cell line (S281E) shows a decrease in thenumber of clusters, compared with the wild-type cell line.

Therefore, this result showed that, when serine 281 of mouse OCT4(serine 288 of human OCT4) was phosphorylated, the stemness of the stemcells was repressed.

Example 2. Confirmation of Relationship Between Methylation of OCT4Protein and OCT4 Activation

2-1. Identification of OCT4 Methylation in Mouse Embryonic Stem Cells

To confirm a new methylation site of OCT4, following the isolation andpurification of OCT4 protein in mouse embryonic stem cells, analysis wascarried out using a mass spectrometer.

First, to identify the OCT4 protein, a mouse embryonic stem cell line inwhich the FLAG fusion OCT4 protein, instead of endogenous OCT4, isexpressed at the same level as endogenous OCT4 was produced. Then, inthe cell line, only the OCT4 protein was identified using an anti-FLAGprotein antibody through immunoprecipitation, and the result is shown inFIG. 3.

Afterward, the analysis with a mass spectrometer showed that, as shownin FIG. 4, new methylation occurs at the arginine 179 site of mouse OCT4(the arginine 186 site of human OCT4) and the lysine 215 site of mouseOCT4 (the lysine 222 site of human OCT4), which were previously unknown.

2-2. Confirmation of Relationship Between OCT4 Methylation and OCT4Activation

The relationship between OCT4 methylation and OCT4 activation can beconfirmed by a method of evaluating maintenance of embryonic stem cells.For evaluation, first, as shown in FIG. 5, methylation-inhibited mutantsR179K and K215R in which arginine 179 of OCT4 was replaced with lysineor lysine 215 of OCT4 was replaced with arginine were produced throughsite-directed mutagenesis. Afterward, OCT4 in embryonic stem cells wasremoved, and new cell lines expressing normal OCT4 or mutants wereproduced. For cell production, a retrovirus vector expressing wild-typeOCT4 and a puromycin-resistant gene, or OCT4 substituted with OCT4methylation-inhibited R179A/K215A and a puromycin-resistant gene wasproduced using a pMSCV-FLAG-puro vector, and then introduced, fortransfection, into a mouse embryonic stem cell line ZHBTc4, which caninhibit the expression of endogenous OCT4 by treatment with doxycycline.And then, a medium was treated with puromycin to isolate a normallytransfected cell line, and the isolated cell line was treated withdoxycycline to inhibit the expression of endogenous OCT4 and to allowthe substituted exogenous OCT4 protein to be expressed. Afterward, toconfirm whether the cells were transfected with the same amount ofretroviruses, a mouse fibroblast cell line NIH3T3 in which OCT4 was notexpressed was also transfected. The protein expression was confirmedusing OCT4 antibody (Santa Cruz) through western blotting, and as acontrol, ACTB antibody (Abcam) was used.

As a result, as shown in FIG. 6, it was demonstrated that OCT4substituted with methylation-inhibited R179A/K215A was expressed at asimilar level to the wild type (WT) in the NIH3T3 cell line.

In addition, before treatment with doxycycline, the cell line was washedwith PBS, cell clusters were dissociated into single cells usingtrypsin-EDTA (TE) and then a cell culture medium was added so that thecell lysate had a final volume of 1 mL. Afterward, the cell lysate wasdispensed into a 6-well plate at 100 μL, that is, 1/10 of the totalvolume of the cell lysate, treated with doxycycline and puromycin, andcultured at 37° C. in 5% CO₂ for 5 to 7 days. The cultured cells werestained using an alkaline phosphatase assay kit (Medsource OzoneBiomedicals), and observed using a microscope.

As a result, as shown in FIG. 7, it was demonstrated that the OCT4methylation-inhibited mutant cell line shows a decrease in the number ofembryonic stem cells forming clusters, compared with the wild-type cellline as the control.

Therefore, this result showed that, when the methylation of arginine 179of mouse OCT4 (arginine 186 of human OCT4) or lysine 215 of mouse OCT4(lysine 222 of human OCT4) is inhibited, the stemness of the stem cellsis inhibited.

Example 3. Confirmation of Relationship Between Phosphorylation ofSerine 351 of Mouse OCT4 and OCT4 Activation

3-1. Construction of OCT4-Phosphorylated Antibody

To produce an antibody which can specifically recognize only OCT4phosphorylation, an OCT4 protein sequence was provided from the NationalCenter for Biotechnology Information (NCBI), the sequence of 14 aminoacids with OCT4 phosphorylation was screened as shown in FIG. 8, andthen antibody production was referred to an antibody production company(GenScript Biotech Corp., New Jersey, United States).

Subsequently, to confirm the produced antibody, the embryonic stem cellline was washed with PBS, the cells were lysed with a lysis buffer (20mM Tris-Cl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% (v/v) Triton X-100 andProtease inhibitor (#78415, Thermo Scientific)), and then identified bySDS-PAGE and western blotting using the produced antibody, OCT4 antibody(#sc-5279, Santa Cruz) and ACTB antibody (#ab8227, Abcam).

To confirm whether the OCT4 phosphorylation at the serine 351 site isspecifically recognized, it was confirmed whether the recognition isinhibited when the produced antibody is recognized along with a peptidehaving a phosphorylated site during western blotting for a protein ofthe embryonic stem cell line. As a result, as shown in FIG. 9, it wasdemonstrated that, when the peptide having the phosphorylated site wasrecognized by the antibody, the phosphorylation of serine 351 of OCT4was not recognized, and when the antibody was not recognized along withthe peptide or recognized along with a peptide having a site rather thanthe phosphorylated site, phosphorylation was recognized.

In addition, as a result of confirming whether OCT4 or OCT4phosphorylation was recognized depending on the presence or absence ofdoxycycline in the embryonic stem cell line which was treated withdoxycycline to artificially inhibit the expression of endogenous OCT4,as shown in FIG. 10, it was confirmed that, when doxycycline wastreated, OCT4 phosphorylation was not recognized, and when doxycyclinewas not treated, OCT4 phosphorylation was recognized.

In addition, it was confirmed whether OCT4 phosphorylation wasrecognized by the antibody produced as described above throughimmunofluorescence. To this end, an embryonic stem cell line was washedwith PBS, the cells were fixed with 4% formaldehyde (#F1635, Sigma),washed again with PBS, and treated with 0.5% Triton X-100 (#069,Amresco) to increase permeability of a cell membrane. Moreover, thereaction was blocked with 1% bovine serum albumin (BSA, #A0100-050,GeneDEPOT), and then a peptide having a phosphorylated site and apeptide having a site other than the phosphorylated site were reactedwith an OCT4-phosphorylated antibody at room temperature for 2 hours.Afterward, a non-binding antibody was removed with PBS, afluorescence-binding secondary antibody (IgG-FITC, #31569, Thermo FisherScientific) was treated to allow a reaction at room temperature for 1hour, a non-binding antibody was removed again with PBS, and a nucleuswas stained with 4′,6-diamidino-2-phenylindole (DAPI, #CAS 28718-90-3,Calbiochem). In addition, a mounting solution was added to the cells,and protein expression was confirmed using a confocal microscope. As aresult, as shown in FIG. 11, it was demonstrated that when the peptidehaving the phosphorylated site was recognized along with the antibody,the OCT4 phosphorylation at serine 351 was not recognized, and when apeptide having a site other than the phosphorylated site was alsorecognized, phosphorylation was recognized.

In addition, to confirm whether the recognized OCT4 phosphorylation wasderived from OCT4 protein, immunoprecipitation was performed. First, theOCT4 protein was precipitated using OCT4 antibody in the cell lysate ofthe embryonic stem cell line, and the result was confirmed by westernblotting with an OCT4-phosphorylated antibody. Consequently, as shown inFIG. 12, it was confirmed that the recognized OCT4 phosphorylation wasderived from the OCT4 protein.

3-2. Confirmation of OCT4 Phosphorylation in Human Cancer Cells

It was confirmed whether the phosphorylation of serine 351 of OCT4recognized in Example 3-1 was detected in human cancer cells as well asthe embryonic stem cell line. To this end, NCCIT and NTERA2 cell lineshaving characteristics of the embryonic stem cells among human cancercells were used, mouse embryonic stem cells (E14), as a positivecontrol, and human fetal kidney stem cells (293FT), as a negativecontrol, were used. Through western blotting described in Example 3-1,as shown in FIG. 13, it was observed that OCT4 phosphorylation wasdetected in human cancer cells.

3-3. Confirmation of Relationship Between OCT4 Phosphorylation and OCT4Activation

The relationship between OCT4 phosphorylation and OCT4 activation can bedetermined by a method of evaluating maintenance of the embryonic stemcells. To this end, first, a phosphorylation-inhibited mutant S351A inwhich serine 351 of OCT4 was replaced with alanine and aphosphorylation-mimetic mutant S351E in which serine 351 of OCT4 wasreplaced with glutamic acid were produced through site-directedmutagenesis. Afterward, OCT4 in the embryonic stem cells was removed,and new cell lines exhibiting normal OCT4 or OCT4 mutants were produced.For cell production, a retrovirus vector expressing wild-type OCT4 and apuromycin-resistant gene, or OCT4 substituted with OCT4phosphorylation-inhibited/mimetic S351A/S351E and a puromycin-resistantgene was produced using a pMSCV-FLAG-puro vector, and then introduced,for transfection, into a mouse embryonic stem cell line ZHBTc4, whichcan inhibit the expression of endogenous OCT4 by treatment withdoxycycline. And then, a medium was treated with puromycin to isolate anormally transfected cell line, and the isolated cell line was treatedwith doxycycline to inhibit the expression of endogenous OCT4 and toallow the substituted exogenous OCT4 protein to be expressed. To confirmwhether the cells were transfected with the same amount of retroviruses,a mouse fibroblast cell line NIH3T3 in which OCT4 was not expressed wasalso transfected. Protein expression was confirmed through westernblotting using OCT4 antibody (#sc-5279, Santa Cruz), and as a control,ACTB antibody (#ab8227, Abcam) was used.

As a result, as shown in FIG. 14, it was demonstrated that OCT4substituted with phosphorylation-inhibited/mimetic S351A/S351E wasexpressed at a similar level to the wild type (WT) in the NIH3T3 cellline.

In addition, before treatment with doxycycline, the cell line was washedwith PBS, cell clusters were dissociated into single cells usingtrypsin-EDTA (TE; Ser. No. 15/400,054, Thermo Fisher) and then a cellculture medium was added so that the cell lysate had a final volume of 1mL. Afterward, the cell lysate was dispensed into a 6-well plate at 100μL, that is, 1/10 of the total volume of the cell lysate, treated withdoxycycline and puromycin, and cultured at 37° C. in 5% CO₂ for 5 to 7days. The cultured cells were stained using an alkaline phosphataseassay kit (Medsource Ozone Biomedicals), and observed using amicroscope.

As a result, as shown in FIG. 15, it was demonstrated that the number ofembryonic stem cells forming clusters in the OCT4phosphorylation-inhibited mutant cell line (S351A), compared with thewild-type cell line as a control was decreased, whereas the number ofclusters was restored in the OCT4 phosphorylation-mimetic mutant cellline (S351E) to as much as in the wild-type cell line.

In addition, a cell line stably expressing an OCT4 mutant protein wasproduced, and an effect of the OCT4 phosphorylation on the embryonicstem cells was confirmed. To this end, a recombinant plasmid expressingwild type OCT4 and a puromycin-resistant gene, or OCT4 substituted withOCT4 phosphorylation-inhibited/mimetic S351A/S351E and apuromycin-resistant gene was produced using a pCAG-Flag vector, andintroduced, for transfection, into a mouse embryonic stem cell lineZHBTc4 inhibiting the expression of endogenous OCT4 by treatment withdoxycycline using Lipofectamine 3000 (Thermo Fisher). Afterward, themedium was treated with puromycin to normally isolate the transfectedcell line, the isolated cell line was treated with doxycycline toinhibit the expression of endogenous OCT4, and then exogenous OCT4protein-substituted cell lines (ZHBTc4 OCT4-WT, ZHBTc4 OCT4-S351A andZHBTc4 OCT4-S351E) were produced. The produced cell lines wereidentified by western blotting. The cell lines cultured for 4 to 8 daysafter treatment with doxycycline were washed with PBS, the cells werelysed with a lysis buffer (20 mM Tris-Cl (pH 7.4), 150 mM NaCl, 1 mMEDTA, 1% (v/v) Triton X-100 and protease inhibitors), and then subjectedto SDS-PAGE and western blotting using an OCT4 antibody and a FLAGantibody (#F3165, Sigma). As a control, an ACTB antibody was used.

As a result, as shown in FIG. 16, it was demonstrated that wild-typeOCT4 or S351A/5351E-substituted OCT4 was normally transformed, and theexogenous OCT4 protein was doxycycline-dependently expressed as much asthe endogenous OCT4 protein.

In addition, the produced cell line was divided into single cells, and1000 single cells were dispensed into each well of a 6-well plate andthen cultured at 37° C., in 5% CO₂ for 5 to 7 days while treatingdoxycycline and puromycin. The cultured cells were stained using analkaline phosphatase assay kit (Medsource Ozone Biomedicals), andobserved using a microscope.

As a result, as shown in FIG. 17, it was demonstrated that the cellsstably expressing phosphorylation-inhibited OCT4 (S351A), compared withthe wild type as a control, had widely spread cell morphology, whereasthe cells expressing phosphorylation-mimetic OCT4 (S351E) had adome-shaped cell morphology like the control.

In addition, the self-renewal ability of stem cells was evaluated usinga SSEA-1 antibody as a marker for self-renewal ability of a stem cell.To this end, 1000 single cells were dispensed into each well of a 6-wellplate, cultured at 37° C. in 5% CO₂ for 5 to 7 days while treatingdoxycycline and puromycin, stained with SSEA-1 and identified byfluorescence activated cell sorting (FACS).

As a result, as shown in FIG. 18, the cell line expressingphosphorylation-mimetic OCT4 (S351E), that is, ZHBTc4 OCT4-S351E, wasstained with SSEA-1 like the wild type (ZHBTc4 OCT4-WT), whereas thecell line expressing phosphorylation-inhibited OCT4 (S351A), that is,ZHBTc4 OCT4-S351A, was less stained.

Example 4. Confirmation of Relationship Between OCT4 Phosphorylation andRegulation of Wnt Signaling

4-1. Evaluation of Effect of OCT4 Phosphorylation on Embryonic StemCells

The embryonic stem cell lines (ZHBTc4 OCT4-WT and ZHBTc4 OCT4-S351A)stably expressing exogenous OCT4, which were produced by the methoddescribed in Example 3-3 were treated with doxycycline, total RNA waspurified from the cultured cells using a RNeasy Purification Kit(#74104, Qiagen) and then subjected to total RNA-sequencing, conductedby Macrogen (Korea). Through the total RNA-sequencing, a gene fordirectly regulating OCT4 using known ChIP data was screened. The resultis shown in FIG. 19.

A pathway analysis for the gene screened thereby was carried out, andthe result was visualized using a visualization program (Cytoscape,3.6.1). According to the pathway analysis, as shown in FIG. 20, it wasconfirmed that there were changes in a pathway related to thepluripotency of stem cells and a Wnt signaling pathway. Particularly, itwas confirmed that, in the Wnt signaling pathway, whenphosphorylation-inhibited mutant OCT4 was expressed, Wnt signaling wasinhibited through Gene Set Enrichment Analysis (GSEA), and the result isshown in FIG. 21.

4-2. Confirmation of Relationship Between OCT4 Phosphorylation and WntSignaling

The produced cell line produced by the same method as described inExample 3-3 was divided into single cells, and 1000 single cells weredispensed into each well of a 6-well plate and then cultured at 37° C.,in 5% CO₂ for 5 to 7 days while treating doxycycline, puromycin, andrecombinant protein WNT3A or compound CHIR9901, which can artificiallyactivate Wnt signaling. The cultured cells were stained using analkaline phosphatase assay kit, and observed using a microscope.

As a result, as shown in FIG. 22, it was demonstrated that, when Wntsignaling was artificially activated in the cells stably expressingphosphorylation-inhibited OCT4, the self-renewal ability of stem cellswas restored.

In addition, the self-renewal ability of stem cells was evaluated usinga self-renewal ability of stem cells, that is, SSEA-1 antibody, andtherefore, as shown in FIG. 23, it was confirmed that, when the Wntsignaling was activated, the self-renewal ability was restored.

Example 5. Confirmation of OCT4 Phosphorylation-Inducing Kinase

To confirm the relationship between CSNK2A1 and OCT4 phosphorylationusing a kinase inducing the phosphorylation of serine 351 of OCT4, acell line for regulating the inhibition of CSNK2A1 expression wasproduced. To this end, a lentivirus doxycycline-dependently expressingshRNA of SEQ ID NO: 5 or 6 was produced and transfected into anembryonic stem cell line E14, thereby producing a cell line capable ofregulating CSNK2A1 expression using doxycycline. Afterward, followingwestern blotting performed by the same method as described in Example3-1, as shown in FIG. 24, it was confirmed that a kinase inducing OCT4phosphorylation is CSNK2A1.

In addition, an in vitro kinase assay was performed using a GST fusionrecombinant OCT4 protein and GST fusion recombinant CSNK2A1. 1 μg of theGST fusion OCT4 recombinant protein and 0.2 μg of the GST fusion CSNK2A1recombinant protein were mixed with 0.5 mM ATP in a kinase buffer (60 mMHEPES-NaOH pH 7.5, 3 mM MgCl₂, 3 mM MnCl₂, 3 μM Na-orthovanadate, 1.2 mMDTT) to allow a reaction at 30° C. for 30 minutes, followed by SDS-PAGEand western blotting. As a result, as shown in FIG. 25, it wasdemonstrated that, in the presence of OCT4 and CSNK2A1, OCT4phosphorylation was recognized by a phosphorylated antibody.

From the above-described examples, it was confirmed that the OCT4modification-regulating material of the present invention regulates OCT4modification (post-translational modification), thereby regulating theself-renewal ability of stem cells, and the result is schematicallyshown in FIG. 26.

It would be understood by those of ordinary skill in the art that theabove descriptions of the present invention are exemplary, and theexemplary embodiments disclosed herein can be easily modified into otherspecific forms without changing the technical spirit or essentialfeatures of the present invention. Therefore, it should be interpretedthat the exemplary embodiments described above are exemplary in allaspects, and are not limitative.

INDUSTRIAL APPLICABILITY

As the OCT4 modification-regulating material according to the presentinvention regulates OCT4 phosphorylation or methylation and inhibits Wntsignaling, the stemness of various stem cells can be effectivelydegraded, and therefore the OCT4 modification-regulating material can beeffectively used in inhibition of proliferation, recurrence andmetastasis of cancer, and resistance to an anticancer agent.

1. A method of inhibiting stemness through regulation of OCT4modification in vitro, comprising any one or mere selected from thegroup consisting of the following: (a) phosphorylating serine 281 in theamino acid sequence of mouse octamer-binding transcription factor 4(OCT4) or serine 288 in the amino acid sequence of human OCT4: (b)inhibiting the methylation of arinine 179 in the amino acid sequence ofmouse OCT4, arginine 186 in the amino acid sequence of human OCT4,lysine 215 in the amino acid sequence of mouse OCT4, or lysine 222 inthe amino acid sequence of human OCT4; and (c) inhibiting thephosphorylation of Serine 351 in the amino acid sequence of mouse OCT4or serine 359 in the amino acid sequence of human OCT4.
 2. The methodaccording to claim 1, further comprising: in the inhibiting (c),blocking the activation of Wnt signaling.
 3. The method according toclaim 1, wherein the amino acid sequence of mouse OCT4 is represented byan amino acid sequence of SEQ ID NO:
 1. 4. The meth according to claim1, wherein the amino acid sequence of human OCT4 is represented by anamino acid sequence of SEQ ID NO:
 2. 5. The method according to claim 1,wherein the stem cells are any one selected from the group consisting ofembryonic stem cells, gametes, and cancer stem cells.
 6. A method ofregulating stemness through regulation of the function of OCT4modification in vitro, comprising: inhibiting the phosphorylation ofserine 351 in the amino acid sequence of mouse octamer-bindingtranscription factor 4 (OCT4) or serine 359 in the amino acid sequenceof human OCT4; and activating Wnt signaling.
 7. An antibody fordetecting whether serine 351 in the amino acid sequence of mouseoctamer-binding transcription factor 4 (OCT4) or serine 359 in the aminoacid sequence of human OCT4 is phosphorylated, wherein the antibodyrecognizes an epitope represented by an amino acid sequence of SEO IDNO: 7 or 8 and specifically binds to phosphorylated serine 351 of mouseOCT4 or phosphorylated serine 359 of human OCT4.
 8. A method ofscreening a material for repressing the stemness of stem cells,comprising: (a) treating cells expressing mouse octamer-bindingtranscription factor 4 (OCT4) or human OCT4 with a candidate material;(b) detecting any one or more selected from the group consisting of thephosphorylation of serine 281 in the amino acid sequence of mouse OCT4or serine 288 in the amino acid sequence of human OCT4, the methylationof arginine 179 in the amino acid sequence of mouse OCT4, arginine 186in the amino acid sequence of human OCT4, lysine 215 in the amino acidsequence of mouse OCT4, or lysine 222 in the amino acid sequence ofhuman OCT4, and the phosphorylation of serine 351 in the amino acidsequence of mouse OCT4 or serine 359 in the amino acid sequence of humanOCT4; and (c) selecting the candidate material as a material forrepressing the stemness of stem cells when serine 281 in the amino acidsequence of mouse OCT4 or serine 288 in the amino acid sequence of humanOCT4 is phosphorvlated, when the methylation of arginine 179 in theamino acid sequence of mouse OCT4, arginine 186 in the amino acidsequence of human OCT4, lysine 215 in the amino acid sequence of mouseOCT4, or lysine 222 in the amino acid sequence of human OCT4 isinhibited, or when the phosphorylation of serine 351 in the amino acidsequence of mouse OCT4 or serine 359 in the amino acid sequence of humanOCT4 is inhibited.
 9. The method according to claim 8, comprising:detecting whether Wnt signaling is activated in cells as the detecting(b); and selecting the candidate material as a material for repressingthe sternness of stem cells when the Wnt signaling is not activated asthe selecting (c).
 10. A method of diagnosing whether the stemness ofstem cells is repressed, comprising: (a) detecting any one or moreselected from the group consisting of the phosphorylation of serine 281in the amino acid sequence of mouse octamer-binding transcription factor4 (OCT4) or serine 288 in the amino acid sequence of human OCT4, themethylation of arginine 179 in the amino acid sequence of mouse OCT4,arginine 186 in the amino acid sequence of human OCT4, lysine 215 in theamino acid sequence of mouse OCT4, or lysine 222 in the amino acidsequence of human OCT4, and the phosphorylation of serine 351 in theamino acid sequence of mouse OCT4 or serine 359 in the amino acidsequence of human OCT4; and (b) determining that the stemness of stemcells is repressed when serine 281 in the amino acid sequence of mouseOCT4 or serine 288 in the amino acid sequence of human OCT4 isphosphorylated, when the methylation of arginine 179 in the amino acidsequence of mouse OCT4, arginine 186 in the amino acid sequence of humanOCT4, lysine 215 in the amino acid sequence of mouse OCT4, or lysine 222in the amino acid sequence of human OCT4 is inhibited, or when thephosphorylation of serine 351 in the amino acid sequence of mouse OCT4or serine 359 in the amino acid sequence of human OCT4 is inhibited. 11.The method according to claim 10, comprising: confirming whether Wntsignaling is activated in cells expressing mouse OCT4 or human OCT4 asthe detecting (a); and determining that the stemness of stem cells isrepressed when the Wnt signaling is not activated as the determining(b). 12-29. (canceled)